Transformed with 2ul of DNA on 15ul bacteria; twice for each plasmid. Took 3 ul from each incubation of and added into two two mixed SOC cultures. Let's see what happens when LacI GFP, RFP, YFP are co-cultured.

One of each LacI plasmid type, GFP, RFP, YFP, LacI were cultured overnight on plates treated with 10mM IPTG.

Microscope Notes

Overlap between FITC and YGFP blocks prevents differentiation between YFP and GFP. RFP can be differentiated between YFP/GFP very well.

YFP/GFP fades ~3 hours after being taken out of the incubator after overnight incubation. RFP was still strong at +3h.

All 2x DNA vacuum centrifuged. All in 20ul of top10F' competant cells.

Ligation

8ul KaiA + 2ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase

8ul KaiB + 2ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase

8ul KaiC + 2ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase

5ul KaiA + 5ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase

5ul KaiB + 5ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase

5ul KaiC + 5ul J04500 (2x?) + 10ul Ligation Buf + 1ul Roche ligase

2ul KaiA + 8ul J04500 + 10ul Ligation Buf + 1ul Roche ligase

2ul KaiB + 8ul J04500 + 10ul Ligation Buf + 1ul Roche ligase

2ul KaiC + 8ul J04500 + 10ul Ligation Buf + 1ul Roche ligase

8ul GP KaiA + 2ul J04500 + 10ul Ligation Buf + 1ul Roche ligase

8ul GP KaiB + 2ul J04500 + 10ul Ligation Buf + 1ul Roche ligase

8ul GP KaiC + 2ul J04500 + 10ul Ligation Buf + 1ul Roche ligase

From each ligation, two plates were made:

2ul ligation mix + 14ul Top10F' cells

16ul ligation mix + 14ul Top10F cells.

Samples 10, 11, and 12 were plated on Carb plates, as gel purification removes the need to select against uncut GeneArt plasmids

Results

12 hours later, no Kan plates had colonies except for the positive control.

12 hours later, all carb plates except for the 2ul #12 plate had colonies. The Carb positive control had an prder of magnitude more cells than the experimental plates. Experimental plates had between 500 and 1000 colonies. Contamination from uncut GeneArt plasmid is feared. However, there is reason to believe that the Carb plates would do better as the Top10f' kit describes Kanomycin selection as "decreasing" transformation efficiency.

The colonies seen on the Carb plates were digested, re-ligated GeneArt plasmid. The gel purification did not eliminate all cut backbone, and the Carb selection did not remove the religated form.

Many colonies from 8.30 AMP plates #10 #11 and #12 were restreaked on Kan plates, selecting against re-ligated GeneArt plasmids in hopes of identifying an insert-holding plasmid.

Result: two different streaks produced colonies for both #10 (KaiA) and #11 (KaiB). A colony PCR was started for 4 colonies of each streak. Noteto self: KaiA and KaiB labels switched on PCR and in LB cultures. Label A2.4 may have mixed lb culture.

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To Sequence:

8.31 Restreak KaiA 1.1 (Putative KaiA + BB)

8.31 Restreak KaiA 2.4 (Putative KaiA + BB)

8.31 Restreak KaiB 1.1 (Putative KaiB + BB)

8.31 Restreak KaiB 2.1 (Putative KaiB + BB)

9.2 KaiB Colony 1 (Putative KaiB + J04500 + BB)

9.2 8.31 #6 (11) Colony 2 (Putative KaiB + BB)

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To Miniprep:

8.31 Restreak KaiA 1.1 (Putative KaiA + BB)

8.31 Restreak KaiA 1.3 (Putative KaiA + BB)

8.31 Restreak KaiB 2.1 (Putative KaiA + BB)

8.31 Restreak KaiB 2.4 (Putative KaiA + BB)

9.2 KaiA Colony 2 (Putative KaiB + J04500 + BB)

9.2 KaiA Colony 3 (Putative KaiB + J04500 + BB)

9.2 KaiA Colony 6 (Putative KaiB + J04500 + BB)

9.2 KaiA Colony 8 (Putative KaiB + J04500 + BB)

9.2 KaiB Colony 2 (Putative KaiB + J04500 + BB)

9.2 KaiB Colony 3 (Putative KaiB + J04500 + BB)

9.2 KaiB Colony 6 (Putative KaiB + J04500 + BB)

9.2 KaiB Colony 8 (Putative KaiB + J04500 + BB)

9.2 KaiC Colony 2 (Putative KaiB + J04500 + BB)

9.2 KaiC Colony 3 (Putative KaiB + J04500 + BB)

9.2 KaiC Colony 5 (Putative KaiB + J04500 + BB)

9.2 KaiC Colony 6 (Putative KaiB + J04500 + BB)

And the results...

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Results Summary

Putative ligation product identities

A09 - J04500 + KaiA

A12 - J04500 + KaiA

A16 - J04500 + KaiA

A18 - J04500 + KaiA

A20 - J04500 + KaiA

B11 - J04500 + KaiB

B18 - J04500 + KaiB

C16 - J04500 + KaiC

C19 - J04500 + KaiC

Each strain is currently streaked on a Kan plate; glycerols and sequencing pending.